Search

The fibroblast growth factor receptor 2 ( FGFR 2) gene is one of the most extensively studied genes with many known mutations implicated in several human disorders, including oncogenic ones. Most FGFR2 disease-associated gene mutations are missense mutations that result in constitutive activation of the FGFR2 protein and downstream molecular pathways. Many tertiary structures of the FGFR2 kinase domain are publicly available in the wildtype and mutated forms and in the inactive and activated state of the receptor. The current literature suggests a molecular brake inhibiting the ATP-binding A loop from adopting the activated state. Mutations relieve this brake, triggering allosteric changes between active and inactive states. However, the existing analysis relies on static structures and fails to account for the intrinsic structural dynamics. In this study, we utilize experimentally resolved structures of the FGFR2 tyrosine kinase domain and machine learning to capture the intrinsic structural dynamics, correlate it with functional regions and disease types, and enrich it with predicted structures of variants with currently no experimentally resolved structures. Our findings demonstrate the value of machine learning-enabled characterizations of structure dynamics in revealing the impact of mutations on (dys)function and disorder in FGFR2.

Exome and genome sequencing are increasingly utilized in research studies and clinical care and can provide clinically relevant information beyond the initial intent for sequencing, including medically actionable secondary findings. Despite ongoing debate about sharing this information with patients and participants, a growing number of clinical laboratories and research programs routinely report secondary findings that increase the risk for selected diseases. Recently, there has been a push to maximize the potential benefit of this practice by implementing proactive genomic screening at the population level irrespective of medical history, but the feasibility of deploying population-scale proactive genomic screening requires scaling key elements of the genomic data evaluation process. Herein, we describe the motivation, development, and implementation of a population-scale variant-first screening pipeline combining bioinformatics-based filtering with a manual review process to screen for clinically relevant findings in research exomes generated through the DiscovEHR collaboration within Geisinger's MyCode® research project. Consistent with other studies, this pipeline yields a screen-positive detection rate between 2.1 and 2.6% (depending on inclusion of those with prior indication-based testing) in 130,048 adult MyCode patient-participants screened for clinically relevant findings in 60 genes. Our variant-first pipeline affords cost and time savings by filtering out negative cases, thereby avoiding analysis of each exome one-by-one, as typically employed in the diagnostic setting. While research is still needed to fully appreciate the benefits of population genomic screening, MyCode provides the first demonstration of a program at scale to help shape how population genomic screening is integrated into routine clinical care., (© 2021 Wiley Periodicals LLC.)

In the version of this article published, the P values for the enrichment of single mutation categories were inadvertently not corrected for multiple testing. After multiple-testing correction, only two of the six mutation categories mentioned are still statistically significant. To reflect this, the text "More specifically, paternally derived DNMs are enriched in transitions in A[.]G contexts, especially ACG>ATG and ATG>ACG (Bonferroni-corrected P = 1.3 × 10 -2 and P = 1 × 10 -3 , respectively). Additionally, we observed overrepresentation of ATA>ACA mutations (Bonferroni-corrected P = 4.28 × 10 -2 ) for DNMs of paternal origin. Among maternally derived DNMs, CCA>CTA, GCA>GTA and TCT>TGT mutations were significantly overrepresented (Bonferroni-corrected P = 4 × 10 -4 , P = 5 × 10 -4 , P = 1 × 10 -3 , respectively)" should read "More specifically, CCA>CTA and GCA>GTA mutations were significantly overenriched on the maternal allele (Bonferroni-corrected P = 0.0192 and P = 0.048, respectively)." Additionally, the last sentence to the legend for Fig. 3b should read "Green boxes highlight the mutation categories that differ significantly" instead of "Green boxes highlight the mutation categories that differ more than 1% of mutation load with a bootstrapping P value <0.05." Corrected versions of Fig. 3b and Supplementary Table 25 appear with the Author Correction.

De novo mutations (DNMs) originating in gametogenesis are an important source of genetic variation. We use a data set of 7,216 autosomal DNMs with resolved parent of origin from whole-genome sequencing of 816 parent-offspring trios to investigate differences between maternally and paternally derived DNMs and study the underlying mutational mechanisms. Our results show that the number of DNMs in offspring increases not only with paternal age, but also with maternal age, and that some genome regions show enrichment for maternally derived DNMs. We identify parent-of-origin-specific mutation signatures that become more pronounced with increased parental age, pointing to different mutational mechanisms in spermatogenesis and oogenesis. Moreover, we find DNMs that are spatially clustered to have a unique mutational signature with no significant differences between parental alleles, suggesting a different mutational mechanism. Our findings provide insights into the molecular mechanisms that underlie mutagenesis and are relevant to disease and evolution in humans.

This report describes an unbiased method for systematically determining gene function in mammalian cells. A total of 20,704 predicted human full-length cDNAs were tested for induction of the IL-8 promoter. A number of genes, including those for cytokines, receptors, adapters, kinases, and transcription factors, were identified that induced the IL-8 promoter through known regulatory sites. Proteins that acted through a cooperative interaction between an AP-1 and an unrecognized cAMP response element (CRE)-like site were also identified. A protein, termed transducer of regulated cAMP response element-binding protein (CREB) (TORC1), was identified that activated expression through the variant CRE and consensus CRE sites. TORC1 potently induced known CREB1 target genes, bound CREB1, and activated expression through a potent transcription activation domain. A functional Drosophila TORC gene was also identified. Thus, TORCs represent a family of highly conserved CREB coactivators that may control the potency and specificity of CRE-mediated responses.

beta-Secretase (BACE) initiates the amyloidogenic processing of the amyloid precursor protein leading to the generation of the beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE is a type I transmembrane aspartyl protease of 501 amino acids. Here we describe a novel BACE mRNA lacking 132 base pairs that is expressed in the pancreas but not in the brain. Sequence alignment indicates that the deleted fragment matches the terminal two-thirds of exon 3. The new BACE variant is short of a 44-amino acid region located between the two catalytic aspartyl residues. Accordingly, a 50-kDa form of BACE (BACE457) is detected in the human pancreas. When expressed in cells, BACE457 colocalizes with the marker for the endoplasmic reticulum BiP. Moreover, BACE457 remains in a proenzymatic and endoglycosidase H-sensitive state, suggesting that its transport along the secretory pathway is blocked at the level of the endoplasmic reticulum. Notably, this novel form of BACE does not contribute to the processing of the amyloid precursor protein. Our findings suggest that tissue-specific splicing of the BACE mRNA may explain the observation that in the human pancreas robust transcription of the BACE gene does not translate into recovered enzymatic activity.

CD6 is a member of the scavenger receptor cysteine rich protein superfamily (SRCRSF). This family includes many cell surface proteins whose three-dimensional structures and functions are presently not well understood. The extracellular region of CD6 includes 3 SRCR domains. The membrane proximal SRCR domain specifically binds the activated leukocyte cell adhesion molecule (ALCAM), a CD6 ligand belonging to the immunoglobulin superfamily. CD6-ALCAM interactions mediate immune cell adhesion and are implicated in T cell maturation and the regulation of T cell function. On the basis of SRCRSF sequence comparison, a mutagenesis analysis of the membrane proximal SRCR domain of CD6 (CD6D3) has been carried out. Fifteen mutants were characterized. Three CD6 residues were identified in a region of low sequence conservation which, when mutated, abolish ligand binding but not the binding to a panel of conformationally sensitive anti-CD6 mAbs. This study provides the first analysis of residues critical for ligand binding to a member of the SRCRSF.

The Ly-6 superfamily of cell surface molecules includes CD59, a potent regulator of the complement system that protects host cells from the cytolytic action of the membrane attack complex (MAC). Although its mechanism of action is not well understood, CD59 is thought to prevent assembly of the MAC by binding to the C8 and/or C9 proteins of the nascent complex. Here a systematic, structure-based mutational approach has been used to determine the region(s) of CD59 required for its protective activity. Analysis of 16 CD59 mutants with single, highly nonconservative substitutions suggests that CD59 has a single active site that includes Trp-40, Arg-53, and Glu-56 of the glycosylated, membrane-distal face of the disk-like extra-cellular domain and, possibly, Asp-24 positioned at the edge of the domain. The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function. Competition and mutational analyses of the epitopes of eight CD59-blocking and non-blocking monoclonal antibodies confirmed the location of the active site. Additional experiments showed that the expression and function of CD59 are both glycosylation independent.

The structurally related T cell surface molecules CD28 and CTLA-4 interact with cell surface ligands CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APC) and modulate T cell antigen recognition. Preliminary reports have suggested that CD80 binds CTLA-4 and CD28 with affinities (Kd values approximately 12 and approximately 200 nM, respectively) that are high when compared with other molecular interactions that contribute to T cell-APC recognition. In the present study, we use surface plasmon resonance to measure the affinity and kinetics of CD80 binding to CD28 and CTLA-4. At 37 degrees C, soluble recombinant CD80 bound to CTLA-4 and CD28 with Kd values of 0.42 and 4 microM, respectively. Kinetic analysis indicated that these low affinities were the result of very fast dissociation rate constants (k(off)); sCD80 dissociated from CD28 and CTLA-4 with k(off) values of > or = 1.6 and > or = 0.43 s-1, respectively. Such rapid binding kinetics have also been reported for the T cell adhesion molecule CD2 and may be necessary to accommodate-dynamic T cell-APC contacts and to facilitate scanning of APC for antigen.

Background: The T-lymphocyte antigen CD2 is an adhesion molecule implicated in immune responses in vivo. The extracellular regions of the human and rat homologues of CD2 share only 45% sequence identity and bind different protein ligands. Comparison of the human and rat soluble CD2 (sCD2) structures should provide insights into the structural basis of cell surface recognition., Results: We therefore determined the crystal structure of a form of human sCD2 with single N-acetylglucosamine residues at each glycosylation site to 2.5 A resolution with an R-factor of 19.3%. It is composed of two immunoglobulin superfamily domains similar to those of rat sCD2, but the relative orientation of the domains in the two homologues differs by up to 20 degrees. An interaction involving the flat, highly charged, ligand binding GFCC'C" faces of crystallographically related human sCD2 molecules duplicates, in a different lattice, that observed in the rat sCD2 crystals., Conclusions: Intramolecular flexibility appears to be a conserved feature of CD2. The head-to-head interaction between molecules represents a general model for interactions between adhesion molecules of this structural class. Ligand specificity may be influenced by the distribution of charged residues on the binding face.

An equine CD2 cDNA has been isolated by monoclonal antibody screening of a T-lymphocyte cDNA library. The cDNA contained an open reading frame of 1041 bp encoding a translated product of 347 amino acids. Northern blotting analysis revealed a single mRNA species expressed in spleen, thymus and activated peripheral lymphocytes. The predicted amino acid sequence has 50-65% identity with the human, rat and mouse CD2 sequences with greatest similarity shared with the human homologue. Evolutionarily conserved structural and functional domains in CD2 were identified by comparing the sequences of the equine, human, mouse and rat CD2 homologues in the context of the recently derived crystal structure of rat soluble CD2 [Jones, E. Y., Davis, S. J., Williams, A. F., Harlos, K. & Stuart, D. I. (1992) Nature 360, 232-239]. The key conserved features of the extracellular region included core residues necessary to preserve the structural integrity of the molecule, residues in the linker region likely to maintain the unique domain organization of CD2, an array of highly charged residues in the putative ligand-binding face of the molecule and glycosylation-signal distributions that render the putative ligand-binding GFCC'C" face of domain 1 relatively unhindered by glycosylation.

Influenza hemagglutinin (HA) undergoes a conformational change that is required for viral entry. The rearrangement includes exposure of the fusion peptide, a hydrophobic segment buried in the trimer interface of the native protein. Since fusion peptide release triggers the membrane fusion event crucial for viral replication, inhibition of fusion peptide exposure should prevent infection. We reasoned that small molecules that bind to HA and stabilize its nonfusogenic conformation would block viral activity. A computer-assisted method was used to select putative HA ligands. One of the selected compounds, 4A,5,8,8A-tetrahydro-5,8-methano-1,4-naphthoquinone, prevented the conversion of X31 HA to a conformation recognized by alpha-fusion peptide antisera. Several derivatives of this compound, including both benzoquinones and hydroquinones, also showed inhibition. The most effective compounds tested have IC50S between 1 and 20 microM. Representative compounds also inhibited virus-induced syncytia formation, HA-mediated hemolysis, and viral infectivity in vitro. The inhibitors are attractive leads for the development of antiviral drugs and can serve as probes of the mechanism of the conformational change of HA.

An attempt has been made to establish, so far as possible, the specific types of synaptic bulbs on motoneurons which arise from extrinsic (descending and dorsal root) and intrinsic sources (interneurons). This has involved the laborious analysis of thousands of electron micrographs of material from normal motoneuron neuropil, and motoneuron neuropil of animals with lesions of spinal tracts, spinal roots, and motor cortex. Our studies have established that the large synaptic bulbs on dendrites, which are the only type in the spinal cord to possess pre-synaptic synapses (serial synapses), are derived from monosynaptic dorsal root fibers (R bulbs). The presynaptic component of the serial synapse appeared to degenerate at levels below spinal cord transections (P bulbs). By means of transections of spinal cord we have found that descending fibers in the spinal cord terminate as one of at least two distinct classes of synaptic bulbs, one with spheroid synaptic vesicles (S) and one with flattened vesicles (F). There is evidence from several sources that the first type may often be excitatory in function and the second type inhibitory. Only synaptic bulbs with spheroid vesicles show definite signs of degeneration after lesions of the opposite motor cortex. The large (L) synaptic bulbs on motoneuron somata, associated with subsynaptic cisterns, are clearly not derived from the descending systems, or from posterior root fibers.

An electron microscopic atlas of simian cochlear duct structures is presented with the hope that it may help to clarify certain questions of structural-functional relationships. Emphasis is placed on structures directly involved in sensory transduction and in synaptic transmission, at afferent and efferent nerve fiber terminals. A brief discussion is included as an introduction to the possibilities of interpretation of electron microscopic data. Orientation of electron micrographs is assisted by means of numerical coding on light microscopic photographs, and by means of a diagram which combines electron microscopic and light microscopic data.

Presynaptic bodies of auditory hair cells of Old World monkeys are separately differentiated in inner, as contrasted with outer, hair cells. The pre-synaptic bodies of outer cells are spherical and of variable electron density, and are thus similar to those of the labyrinth of vertebrates from fish to man. The difficulty in finding them, as compared with the relative ease of finding the presynaptic bodies of inner hair cells, suggests either that they are not present in all outer hair cells or that they undergo a regression-reconstitution cycle. The presynaptic bodies of simian inner hair cells are almost always ring-shaped. The few exceptions reinforce the impression of a later evolutionary development of the inner hair cell system. In any event, our findings serve to reemphasize the remarkable differentiation of outer and inner hair cell systems, and to deepen the mystery of their separate role in audition.